Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
  • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/84—Brevibacterium
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/843—Corynebacterium

Definitions

• the present invention relates to a method for producing L-lysine by fermentation.
• L-lysine which is used as a feedstuff is normally produced industrially by a fermentation process in which L-lysine producing mutants of the genus Brevibacterium or Corynebacterium are used.
• L-lysine-producing mutants produced by the artificial mutation of wild strains of microorganisms of the genera Brevibacterium and Corynebacterium, are known.
• mutants resistant to S-(2-aminoethyl)-cystein hereinafter identified as AEC
• mutants requiring L-amino acids such as L-homoserine, L-threonine, L-leucine, or L-methionine for their growth
• one object of the present invention is to provide a method of preparing L-lysine in improved yields by fermentation processes.
• L-lysine can be increased when resistance to ethylene glycol is imparted to known L-lysine producing microorganisms of the genus Brevibacterium or Corynebacterium.
• the microorganisms employed in the present invention are mutants which belong to the genus Brevibacterium or Corynebacterium.
• the mutants have the characteristics necessary for the production of L-lysine which are the homoserine requirement, resistance to AEC and resistance to ⁇ -chloro-caprolactam (CCL). Of course, the mutants possess resistance to ethylene glycol.
• Representative mutant specimens of the present invention are:
• AEC r resistance to S-(2-aminoethyl)-L-cystein
• mutants identified above by FERM-BP numbers were originally deposited with the FERM-P numbers on Dec. 27, 1980 at the Fermentation Research Institute, Agency of Industrial Sciences and Technology, Ministry of International Trade and Industry (FRI), 1-3, Higashi 1-chome, yatabe-machi, Tsukuba-gun, Ibaragi-ken 305, Japan and these deposits were converted to deposits under the Budapest Treaty on Dec. 9, 1981 with FRI which has aquired the status of an International Depository Authority as of May 1, 1981.
• the mutants mentioned above can be induced from parent microorganism strains by any conventional method.
• the first step of induction is to mutate the parent strains with a suitable chemical mutagen such as N-methyl-N'-nitro-N-nitrosoguanidine (hereinafter referred to as NG) and nitrous acid or with irradiation by ultraviolet light.
• the second step of the process is to select resistant mutants by picking-up colonies of microorganisms on plates of a nutrient agar medium containing an amount of ethylene glycol which inhibits the growth of the parent strains. Thereafter, the mutants are evaluated for L-lysine productivity by a standard method.
• the ethylene glycol resistant mutants are almost resistant to concentrated aqueous solutions of sodium chloride, potassium chloride, ammonium chloride, ammonium sulfate, potassium sulfate, sodium sulfate, glucose, fructose, sucrose, and maltose which inhibit the growth of the parent strains.
• Suitable parent strains from which the present mutants can be produced include mutants capable of producing L-lysine and wild strains of the genus Brevibacterium or Corynebacterium.
• L-lysine productivity is imparted to the wild strains prior to or after imparting resistance to ethylene glycol to the wild strains.
• resistance to AEC must be imparted or the mutant must possess the homoserine growth requirement.
• the preferred wild strains of the genus Brevibacterium or Corynebacterium are coryne-form glutamic acid producing bacteria and the examples include:
• the microbial cells thus treated were washed with phosphate buffer solution, inoculated on a plate of the minimum culture medium of which the composition is given in Table 1 containing 30 g/dl ethylene glycol, and cultured at 30° C. with shaking for 2 to 4 days until ethylene glycol resistant mutants propagated in the culture medium. Then the culture medium was spread on the agar plates of the minimum culture medium and the plates were incubated at 30° C. for 4 days.
• mutants that were prepared which are within the scope of the present invention include C. acetoglutamicum AJ 11656 and B. flavum AJ 11658. These mutants were obtained by the same culturing process.
• Test strains were washed with the minimum medium and inoculated into the mediums in the test tubes. Then the tubes were incubated at 30° C. for 24 hours with shaking. After the cultivation, the growth of each strain was determined by measuring the optical density at 562 nm of the culture broths. The results are shown in Table 2. The degree of resistance to ethylene glycol is represented by the relative values of the growth to the control.
• the mutants were cultured aerobically in a conventional culture medium containing carbon sources, nitrogen sources, and inorganic ions, and when required, minor nutrients.
• Suitable carbon sources include saccharides such as glucose, fructose, and sucrose, and molasses and hydrolyzed starch containing these saccharides; organic acids such as acetic acid, and propionic acid; and alcohols, which are preferably used.
• Suitable nitrogen sources include, for example, ammonium sulfate, gaseous ammonia and urea.
• Cultivation is carried out preferably under aerobic conditions, for 2 to 7 days at a temperature ranging from 24° to 37° C. with preferable adjustment of the pH of the culture medium to 5.0 to 9.0 with an organic or inorganic acid or alkali.
• urea, CaCO 3 , and gaseous ammonia may be used.
• the L-lysine which accumulates in the culture medium may be recovered by an entirely conventional recovery technique such as those which use an ion-exchange resin.
• a culture broth of AJ 11656 prepared in the same manner as described above was collected and centrifuged to remove microbial cells, and the solid CaCO 3 .
• 1.0 liter supernatant solution thus obtained was passed through a column of "Amberlite IR-120" in the acid form.
• L-lysine was adsorbed on the resin, and it was eluted with 3% ammonia water.
• the eluate was evaporated and the concentrated solution was cooled to a temperature low enough to crystallize L-lysine, and 36.7 g L-lysine.HCl. 2aq crystal was obtained.
• a fifty ml portion of Culture medium B whose composition is given in Table 3, was placed in a 500 ml flask and heated at 110° C. for 4 minutes for sterilization. Then the culture broth was inoculated with B. lactofermentum AJ 11657 which had been been grown on a bouillon agar slant. The inoculated medium was cultured at 31° C. for 18 hours with shaking to prepare a seed culture broth.
• Culture medium D Twenty ml portions of Culture medium D were placed in 500 ml flasks and heated at 110° C. for 5 minutes. Each flask was supplemented with 1.0 g CaCO 3 separately sterilized.

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• Micro-Organisms Or Cultivation Processes Thereof (AREA)

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• 1980
  • 1980-12-29 JP JP55185676A patent/JPS57115186A/ja active Granted
• 1981
  • 1981-12-22 US US06/333,455 patent/US4411997A/en not_active Expired - Lifetime
  • 1981-12-28 FR FR8124304A patent/FR2497231A1/fr active Granted

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